Effects of urea denatureation and pH on the ability of porcine myoglobin to undergo reduction

To determine the effects of globin moiety denaturation and pH on the ability of metmyoglobin (MetMb) to undergo reduction, MetMb isolated from porcine hearts was denatured in 8.5M urea. Both native and denatured MetMb solutions were serially reduced with Na2S2O4 (0, 7.5, 15, 18.75, 22.5, 26.25, 30, 30.75, and 45 umol). Reduction was conducted at pH 5, 5.2, 5.4, 5.6, 6, 6.2, 6.4, 6.6, and 7. After reduction, absorbance was determined at 635 nm and the percent of the original MetMb which was reduced was calculated. The average percent MetMb reduced from the native and denatured forms was 35 and 25%, respectively. pH significantly influenced the percentage of MetMb reduced, especially when pH was <6. If the MetMb was denatured prior to reduction, the influence of pH on its ability to undergo reduction was slight. The percentage of denatured MetMb reduced was higher at pH 7 than at all other pHs. High pH enhanced the ability of MetMb to undergo reduction; while low pH decreases it. Low pH may have denatured the native globin moiety.